PROJECT SUMMARY/ABSTRACT Regulatory T cells (Tregs) are a subset of CD4 T cells that are essential for the maintenance of peripheral tolerance, yet their precise roles during infections remain an active area of investigation. It is well-documented that following certain infections, Tregs are required to attenuate an overly robust immune response to prevent collateral damage to self-tissues. However, we have demonstrated that removal of Tregs prior to infection with Herpes Simplex Virus, type 2 (HSV-2), among other infections, results in delayed clearance of the pathogen, suggesting that the presence of Tregs can be critical to facilitating an appropriately robust and protective immune response. These differing results emphasize that the role played by Tregs during infections is context-dependent, and thus we propose here to focus on the location of the cells and the time post-infection as key factors that influence the role that Tregs play during mucosal virus infection. Recent evidence suggests that there exists a distinct subset of Tregs known as tissue Tregs. These cells have been best-characterized in skin and visceral adipose tissue, where they function to limit inflammation, though it has been suggested that tissue Tregs in other locations function to prevent autoimmunity, to promote fetal and graft tolerance, and to impair anti-tumor immune responses in various non-lymphoid tissues. However, despite the hypothesized role of tissue Tregs in controlling local inflammation to prevent autoimmunity and immunopathology, local immune responses are routinely and beneficially generated against mucosal infections, often without excessive tissue destruction at the infection site, and we thus hypothesize that tissue Tregs are involved in mediating this balance. Additionally, as effector T cell immune memories remain following infection clearance, we hypothesize that regulatory memory also persists such that these tissue memory T cell responses can be controlled under both homeostatic conditions as well as upon pathogen re-encounter to promote local tissue integrity. Therefore, we propose to extend our investigations of the role of Tregs during mucosal virus infection, now with a focus on the presence and consequences of tissue Tregs on anti-viral immune responses in mice and humans. Tissue-resident memory T cells have been intensely studied in recent years, and are now the basis for a promising new vaccine platform, so it is imperative that we understand how such tissue T cell responses might be regulated in order to support tissue protection in the face of a robust immune response.